Fig. 4: RNA-mediated base editing in β-thalassemia HSPCs.

a, b C-G to T-A (a) or A-T to G-C (b) base-editing efficiency, calculated by the EditR software in β-thalassemic erythroblasts subjected to Sanger sequencing. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, peripheral blood-derived non-mobilized HSPCs from 2 donors harboring the CD39/IVS1-110 G > A and CD 8/9 + G/IVS1-110 G > A mutations, respectively). c Frequency of InDels, measured by TIDE analysis, for base-edited samples subjected to Sanger sequencing. Data are expressed as mean ± SEM (n = 2 biologically independent experiments). d CFC frequency for control (transfected with TE buffer) and base-edited samples. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). No statistical differences were observed between control and edited samples (two-way ANOVA). e RT-qPCR analysis of β-like globin mRNA levels in β-thalassemia patient erythroblasts at day 13 of erythroid differentiation. β-like globin mRNA expression was normalized to α-globin mRNA. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). *P = 0.0270; ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). f Analysis of HbF, HbA and HbA₂ by cation-exchange HPLC in β-thalassemia patient RBCs. We calculated the percentage of each Hb type over the total Hb tetramers. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). g Frequency of HbF-expressing cells in the GYPA⁺ population for control and edited samples, as measured by flow cytometry. Data are expressed as mean ± SEM (n = 4 biologically independent experiments, 2 donors). **P = 0.0098 for LRF 8C, or P = 0.0033 for KLF1 (ordinary one-way ANOVA with Dunnett correction for multiple comparisons). Representative flow cytometry histograms showing HbF⁺ cells in GYPA⁺ populations for control and base-edited samples are presented below the graph. h Expression of β-, δ-, ^Gγ-, ^Aγ- and γ- (^Gγ- + ^Aγ-) globin chains measured by RP-HPLC in β-thalassemia patient RBCs. β-like globin expression was normalized to α-globin. The ratio α/non-α globins is reported on top of the graph. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). *P = 0.0148 (two-way ANOVA with Dunnett correction for multiple comparisons). i Analysis of α-globin precipitates by cation-exchange HPLC in β-thalassemia patient RBCs. We calculated the proportion of α-globin precipitates over the total Hb tetramers. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). ***P = 0.0009 for LRF 8C, or P = 0.0004 for KLF1 (one-way ANOVA with Dunnett correction for multiple comparisons). j Frequency of enucleated cells at day 6, 9, 13, 16, and 20 of erythroid differentiation, as measured by flow cytometry analysis of DRAQ5 nuclear staining in control and edited samples. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). Representative flow cytometry histograms showing the DRAQ5^- cell population for unstained, control, and edited samples are presented below the graph. ***P = 0.0003; ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). k Cell size of enucleated cells (DRAQ5^-) at day 13, 16, and 20 of erythroid differentiation, as measured by flow cytometry using the median of forward scatter (FSC) intensity, and normalized to HD RBCs. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). Representative flow cytometry contour plots showing the FSC of DRAQ5^- cell population for control and edited samples are reported below the graph. **P = 0.0033 for D13, or P = 0.0021 for D16, or P = 0.0034 for D20; ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). l–n Frequency of CD36⁺ (l), CD71⁺ (m) and GYPA⁺ (n) cells at day 6, 9, 13, 16, and 20 of erythroid differentiation, as measured by flow cytometry analysis of CD36, CD71, and GYPA erythroid markers. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). Representative flow cytometry histograms showing the CD36⁺ (l), CD71⁺ (m) and GYPA⁺ (n) cell populations for unstained, control, and edited samples are presented below the graph. *P = 0.0241 for CD36/D16, or P = 0.0190 for CD36/D20, or P = 0.0307 for CD71; **P = 0.0072 for CD36, or P = 0.0020 for CD71/LRF 8C, or P = 0.0028 for CD71/KLF1; ***P = 0.0002; ****P ≤ 0.0001 (two-way ANOVA with Dunnett correction for multiple comparisons). o Frequency of α4-Integrin⁺, BAND3⁺ and α4-Integrin⁺/BAND3⁺ in 7AAD^-/GYPA⁺ cells at day 6, 9, 13, 16, and 20 of erythroid differentiation, as measured by flow cytometry analysis of α4-Integrin and BAND3 erythroid markers. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). Representative flow cytometry contour plots showing the α4-Integrin⁺, BAND3⁺ and α4-Integrin⁺/BAND3⁺ cell populations for unstained, control, and edited samples are reported below the graph. p Frequency of apoptotic cells (Annexin V⁺-cells) in control and edited samples at day 13 of erythroid differentiation, as measured by flow cytometry. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). Representative flow cytometry contour plots showing the Annexin V⁺ cell populations for unstained, control, and edited samples are reported on the right side of the graph. **P = 0.0094 for LRF 8C, or P = 0.0014 for KLF1 (one-way ANOVA with Dunnett correction for multiple comparisons). q Frequency of ROS-containing cells (DCFDA⁺ cells) in control and edited samples at day 20 of erythroid differentiation, as measured by flow cytometry analysis in DRAQ⁺ and DRAQ^- cells. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). Representative flow cytometry contour plots showing the DCFDA⁺ cell populations for unstained, control, and edited enucleated samples are reported on the right side of the graph. Source data are provided as a Source Data file.